This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding enzymes involved in methionine metabolism in plants and seeds.
Human food and animal feed derived from many grains are deficient in the sulfur amino acids, methionine and cysteine, which are required in an animal diet. In corn, the sulfur amino acids are the third most limiting amino acids, after lysine and tryptophan, for the dietary requirements of many animals. The use of soybean meal, which is rich in lysine and tryptophan, to supplement corn in animal feed is limited by the low sulfur amino acid content of the legume. Thus, an increase in the sulfur amino acid content of either corn or soybean would improve the nutritional quality of the mixtures and reduce the need for further supplementation through addition of more expensive methionine.
One genetic engineering approach to increase the sulfur amino acid content of seeds is to isolate genes coding for proteins that are rich in the sulfur-containing amino acids methionine and cysteine, to link the genes to strong seed-specific regulatory sequences, to transform the chimeric gene into crops plants and to identify transformants wherein the gene is sufficiently highly expressed to cause an increase in total sulfur amino acid content. However, increasing the sulfur amino acid content of seeds by expression of sulfur-rich proteins may be limited by the ability of the plant to synthesize methionine, by the synthesis and stability of the methionine-rich protein, and by effects of over-accumulation of the methionine-rich protein on the viability of the transgenic seeds.
An alternative approach would be to increase the production and accumulation of the free amino acid, methionine, via genetic engineering technology. However, little guidance is available on the control of the biosynthesis and accumulation of methionine in plants, particularly in the seeds of plants.
Methionine-tRNA ligase (EC 6.1.1.10), also called Methionyl-tRNA synthetase, or L-Methione:tRNA(Met) ligase (AMP-forming) belongs to the class-I aminoacyl-tRNA synthetases. This is a cytoplasmic enzyme which catalyzes the ligation of methionine to transfer RNA in protein translation. Methionine-tRNA ligase is the first enzyme sustaining the methionine pathway in translation initiation in Escherichia coli. This enzyme is probably essential for cell survival, being required not only for elongation of protein synthesis but also for the initiation of all mRNA translation through initiator tRNA-aminoacylation. The active site of methionyl-tRNA synthetase possesses two functions: synthetic, which provides Met-tRNA for protein synthesis, and editing, which rejects inadvertently misactivated homocysteine. During editing, the side chain xe2x80x94SH group of homocysteine reacts with its activated carboxyl group forming a cyclic thioester, homocysteine thiolactone. There is a specific xe2x80x94SH binding subsite, distinct from the methionine binding subsite, in the synthetic/editing active site of methionyl-tRNA synthetase (Jakubowski (1996) Biochemistry 35:8252-8259).
Methionyl-tRNA formyltransferase (10-formyltetrahydrofolate:L-methionyl-tRNA N-transformylase, EC 2.1.2.9) produces formylmethionyl-tRNA. This residue, which already has an amide bond, is the first residue in a polypeptide which initiates with methionine. The structure and/or sequence of the first three base pairs at the end of the amino acid acceptor stem of Escherichia coli initiator tRNA and the discriminator base 73 are important for its formylation by E. coli methionyl-tRNA transformylase. Changes in the rest of the acceptor stem, dihydrouridine stem, anticodon stem, anticodon sequence, and T psi C stem have little or no effect on formylation (Lee et al. (1991) J. Biol. Chem. 266:18012-18017). Corn, Rice and Soybean ESTs encoding polypeptides with similarities to methionyl-tRNA formyltransferase are found in the NCBI database having General Identifier Nos. 485595, 569773, 571553, 3107255, 4292436 4313709, 4573024, 4966952, 5018236, 5055738, 5555461, 5606916, 5649988 and 5666614.
The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 500 amino acids having at least 90% identity, preferably at least 400 amino acids having at least 95% identity, based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn methionyl-tRNA fomyltransferase of SEQ ID NO:2, a rice methionyl-tRNA formyltransferase of SEQ ID NO:4, a soybean methionyl-tRNA formyltransferase of SEQ ID NO:6, a wheat methionyl-tRNA formyltransferase of SEQ ID NO:8, a corn methionyl-tRNA synthetase of SEQ ID NO:10, and a soybean methionyl-tRNA synthetase of SEQ ID NO:12. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above. It is preferred that the isolated polynucleotides of the claimed invention comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9 and 11 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10 and 12. The present invention also relates to an isolated polynucleotide comprising a nucleotide sequences of at least 40 (preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11 and the complement of such nucleotide sequences.
The present invention relates to a chimeric gene comprising an isolated polynucleotide of the present invention operably linked to suitable regulatory sequences.
The present invention relates to an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention. The host cell may be eucaryotic, such as a yeast or a plant cell, or procaryotic, such as a bacterial cell. The present invention also relates to a virus, preferably a baculovirus, comprising an isolated polynucleotide of the present invention or a chimeric gene of the present invention.
The present invention relates to a process for producing an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention, the process comprising either transforming or transfecting an isolated compatible host cell with a chimeric gene or isolated polynucleotide of the present invention.
The present invention relates to a methionyl-tRNA formyltransferase or a methionyl-tRNA synthetase polypeptide of at least 500 amino acids having at least 90% homology, preferably at least 400 amino acids having at least 95% homology, based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10 and 12.
The present invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a methionyl-tRNA formyltransferase or a methionyl-tRNA synthetase polypeptide in a plant cell, the method comprising the steps of:
constructing an isolated polynucleotide or chimeric gene of the present invention;
introducing the isolated polynucleotide into a plant cell;
measuring the level a methionyl-tRNA formyltransferase or a methionyl-tRNA synthetase polypeptide in the plant cell containing the polynucleotide; and
comparing the level of a methionyl-tRNA formyltransferase or a methionyl-tRNA synthetase polypeptide in the plant cell containing the isolated polynucleotide with the level of a methionyl-tRNA formyltransferase or a methionyl-tRNA synthetase polypeptide in a plant cell that does not contain the isolated polynucleotide.
The present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of a methionyl-tRNA formyltransferase or a methionyl-tRNA synthetase gene, preferably a plant methionyl-tRNA formyltransferase or methionyl-tRNA synthetase gene, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least 40 (preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11 and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a portion of a thioredoxin polypeptide amino acid sequence.
The present invention also relates to a method of obtaining a nucleic acid fragment encoding all or a subsantial portion of the amino acid sequence encoding a to a methionyl-tRNA formyltransferase or a methionyl-tRNA synthetase polypeptide comprising the steps of:
probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and
sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.